Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant that plays critical roles in embryogenesis, cell differentiation and wound repair. PDGF is a family of three disulfide-linked glycoprotein dimers of M.sub.r 29,000-32,000 that arise from stochastic assembly of the homologous subunits, A-chain and B-chain, yielding the heterodimer PDGF-AB and homodimers PDGF-AA and PDGF-BB [for a review, see ref. 1]. The A-chain is expressed as two different forms ("long" and "short") as determined by alternative splicing, with the long form containing an extended carboxyl terminus of basic amino acids that mediates association with the extracellular matrix and is encoded by exon 6 [2]. The B-chain also contains a cationic carboxyl terminus that has been implicated in the strong association of PDGF-BB with the cell surface [3]. The activity of PDGF is mediated by two PDGF receptor isotypes, .alpha. and .beta., which upon ligand binding associate noncovalently to form homo- and heterodimers [4,5]. The PDGF .alpha.-receptor has a high affinity for both A- and B-chains, while the .beta.-receptor recognizes only the B-chain [6].
X-ray crystallographic analysis of recombinant human PDGF-BB has revealed an antiparallel arrangement of the disulfide-linked B-chain monomers [7]. Each B-chain monomer is characterized by a so-called "cysteine knot" structure, consisting of three intrachain disulfide bonds and two antiparallel .beta.-strands, interconnected by three loop segments (I-III). The cationic residues Arg.sup.159, Lys.sup.160, Lys.sup.161 of the A-chain, which correspond to the loop III region of the B-chain, are demonstrated as being required for high affinity receptor binding and mitogenic activity [8]. Furthermore, a synthetic peptide containing loop III residues 157-163 of the B-chain exhibits PDGF antagonizing activity [9], while a mutation at residue Lys.sup.161 of the B-chain is shown to interfere with high affinity binding to the .alpha.-receptor [10].
The present invention concerns the transformation of Pichia pastoris with a cDNA encoding the PDGF B chain. Others have used the Pichia pastoris system to express proteins. For instance, U.S. Pat. No. 5,324,639 discloses the expression of insulin-like growth factor-1 in Pichia pastoris cells. The recombinant protein is disclosed as being secreted into the culture medium at the level up to 100 times higher than results published in the literature using Saccharomyces cerevisae as the host cell. However, the reference does not suggest expressing PDGF in this system.
Cook et al., "Purification and Analysis of Proteinase-Resistant Mutants of Recombinant Platelet-Derived Growth Factor-BB Exhibiting Improved Biological Activity," Biochem. J. 281: 57-65 (1992) disclose the expression of recombinant platelet-derived growth factor-BB and two protease-resistant mutants thereof in Saccharomyces cerevisiae. The expression construct utilized sequences encoding the yeast .alpha.-factor pre-pro sequence upstream of the PDGF coding sequences to facilitate secretion. However, secretion into the culture medium was very low and most of the PDGF remained intracellular. The authors state that "a major limiting factor to PDGF-BB expression is, therefore, the secretion of PDGF-BB into the medium."
Craig et al., "Characterization of the Structure and Conformation of Platelet-Derived Growth Factor-BB (PDGF-BB) and Proteinase-Resistant Mutants of PDGF-BB Expressed in Saccharomyces cerevisiae," Biochem. J. 281: 67-72 (1992) disclose structural studies of the recombinant PDGF-BB produced in Cook et al. (above). The authors conclude that the protease-resistant mutants have the same secondary and tertiary structures as the wild-type PDGF-BB. The structure is reported to be considerably different than that reported for authentic PDGF-BB.
Duan et al., "A Functional Soluble Extracellular Region of the Platelet-Derived Growth Factor (PDGF) .beta.-Receptor Antagonizes PDGF-Stimulated Responses," J. Biol. Chem. 266: 413-418 (1991) disclose expression of the PDGFR extracellular domain in Chinese Hamster Ovary cells using an amplifiable marker to increase expression. The publication discloses that the protein was expressed in the culture medium at a level of 12 mg/L. The extracellular domain was extensively glycosylated, and bound to PDGF-BB with an affinity similar to that of the intact PDGF receptor. However, the reference does not teach or suggest expression of a PDGF protein in Pichia pastoris.
There remains a need to increase the efficiency of producing proteins generally, and PDGF proteins in particular. It is expected that with improved production methods the cost of these proteins can be brought down and their availability for treating a wide range of conditions can be increased. PDGF proteins are particularly implicated in as being useful in such applications as cancer therapy, wound healing, and the treatment of gastrointestinal ulcers.